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Abstract

Commensal and pathogenic fungi are exposed to hydrogen peroxide (H2O2) produced by macrophages of the host. Pathogenic fungi counteract the harmful effects of H2O2 with the enzyme catalase (EC 1.11.1.6), which decomposes two molecules of H2O2 to two molecules of H2O and O2. Contribution of antioxidant systems on fungal virulence is actively studied. Measurement of catalase activity can contribute to the elucidation of the factors that influence the regulation of this pivotal enzyme. Here we describe a simple spectrophotometric method in which the activity of catalase is measured in total yeast extracts. Decomposition of H2O2 by the yeast extract is followed by the decrease in absorbance at 240 nm. The difference in absorbance through time (ΔA240) is inferred as the measure of catalase activity.

Keywords: Catalase, Candida, Glabrata, Yeast, CTA1

Materials and Reagents

Yeast strains Note: BG14 was used as the C. glabrata parental strain. The hst1Δ and the cta1Δ null mutants were used as a positive and the negative controls, respectively.

Dilute overnight cultures in 50 ml of fresh medium in order that after seven duplications, the yeast cultures reach an OD600 = 0.5 at 30 °C.

Centrifuge the cells for 5 min at 2,600 x g. Discard supernatant. Temperature of centrifuge is not relevant.

Wash the cells with 25 ml of sterile water and discard supernatant.

Resuspend the cells in 0.5 ml PB with protease inhibitors and transfer to a microfuge tube. Keep the samples on ice.

Add 50 µl of zirconia/silica beads to each sample.

Disrupt the cells by vortexing at maximum speed for 1 min and place on ice for another minute. Repeat 20 times.

Centrifuge the lysate at 25,000 x g for 30 min at 4 °C to remove cell debris and zirconia/silica beads

Transfer supernatant to a clean microfuge tube. At this point, lysates are ready for quantification of total protein and measurement of catalase activity. Alternatively, samples can be stored at -20 °C.

Load 5 µl of the standards and diluted lysates to the Bradford reagent. Incubate room temperature for 5 min.

Measure the absorbance at OD595 in a microplate spectrophotometer.

Determine the amount of protein of the samples from the standard curve. Consider the dilution factor.

Catalase activity assay

Set up the spectrophotometer by first turning on the instrument and then the UV light. Set up a kinetics program to record every 30 s at a wavelength of 240 nm for 2 min.

Calibrate the spectrophotometer using 3 ml of PB in a 3-ml quartz cuvette as a blank.

Dilute the lysate samples 1:50 with PB.

In a quartz cuvette, mix 1 ml PB with 1 ml of the diluted sample. To begin the assay, add 1 ml of the H2O2 solution (H2O2 to a final concentration of 10 mM). The initial absorbance must be between 0.550 and 0.520. If necessary, add H2O2 to increase the absorbance and Phosphate Buffer to decrease the absorbance.

Mix the content by inversion and Immediately place the cuvettes into the spectrophotometer. Follow the decrease in absorbance at OD240 with a stopwatch for 2 min.

A catalase solution must be used as a control. Pippete 2.9 ml of PB in the cuvette, add 1 ml 30 mM H2O2 and 100 µl of the catalase solution (~10 units). Record the initial and final absorbance in a one-minute period. Use 2 ml of PB and 1 ml of 30 mM H2O2 as blank.

Calculate the catalase activity using the following formula (Cuellar-Cruz et al., 2009)
Whereis the difference between the initial and final absorbance. is the total volume of the reaction (3 ml).is the molar extinction coefficient for H2O2 at OD240 (34.9 mol-1 cm-1). is the optical length path of cuvette (1 cm). is the volume of the sample in ml. is the protein concentration of the sample in mg/ml.

Example of catalase activities of extracts from C. glabrata strains in exponential phase of growth:

This protocol is based on the methodology reported by Aebi (1984), and by Weydert and Cullen (2010). Our adapted method was first published in Cuellar-Cruz et al. (2009). This work was funded by a CONACYT grant no. CB-2010-153929 to A.D.L.P. Finally, we thank Guadalupe Gutierrez-Escobedo for technical assistance.

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